The biosynthesis of the iron-molybdenum cofactor of nitrogenase will be studied in the nitrogen-fixing organisms Azotobacter vinelandii and Klebsiella pneumoniae. The Iron-Molybdenum cofactor (FeMo-co) is composed of molybdenum, iron, sulfur and homocitrate in a 1:6-8:4-10:1 ration. The nifQBNEVH genes are required for its synthesis as are several non-nif- specific gene products. The goal of this project will be to completely define the biochemical pathway for FeMo-co biosynthesis and in the process, to deduce the structure of FeMo-co. The enzymes of the FeMo-co biosynthetic pathway will be purified and characterized as to their properties and role in the synthesis. The donors of iron and sulfur to FeMo-co will be identified. Intermediates in the pathway will be isolated and their position in the sequence will be determined. The role of ATP and reductant in FeMo-co synthesis will be tested and the role of the nifH gene product in the synthesis will be studied. The nifH product is dinitrogenase reductase, which also plays a catalytic role in nitrogenase enzymatic activity. Analysis of the pathway will involve use of 99Mo, 55Fe, 35S, and 3H-homocitrate as tracers and 17 O-, 13C- and 19F- homocitrate as well as 95Mo and 57Fe as spectroscopic probes. Protein is a limiting factor in human nutrition, and biological nitrogen fixation is the largest source of fixed nitrogen for protein synthesis on earth. Molybdoenzymes are important in human nutrition and in every biological system. The study of molybdenum metabolism and nitrogen fixation will provide information about how biological systems assimilate and process this important element.